11/14/18 Wednesday ISSR Analysis and Geneious.

I added loading dye into my PCR tubes, which were used from last lab. I added about 1 micro liters to each tubes (15 total).

Then including the solution inside the PCR tube, total of 5 micro liters, I added into the agarose gel that was given by the professor. The first and last wells were saved for ladder. I added 15 of Carter’s samples first and added my samples second. Kayla added her samples and added 3 of Peter’s samples. Last of Peter’s samples (12 samples) were loaded on the lowest line of 50 wells.

After every table loaded their samples onto the wells, professor Paul ran these gels at a low voltage (20 volts) for a long time ans see how things turn out. We didn’t get to see what happened.

Next, we moved on to Geneious. Our table had to do some work with JP5551 because our previous JP5334 was not very good. I did the same thing with JP5551 from the last lab and sent it to professor Paul so that every can download my nucleotide alignment and make concatenated alignment. I made a separate concatenated file and put JP 5334,5551,5525 and 5536 into the file. Then I choose all four documents and choose tools>Concatenate Sequences or Alignments and clicked ok. It created a concatenated alignments and I opened up to see if it looks correct. I have about 1500 nucleotides and I edited/cut the alignment 1029-1085 because it looked bad and thought that it can effect my phylogenetic tree. Next, I exported the file in phylip alignment type and saved it. Then I opened jmodeltest and loaded my alignment and compute likehood scores to calculate AIC and BIC. I got TPM G for both AIC and BIC so for my Bayesian analysis, I chose HKY85, gamma and TG0248. Then ran the analysis for 3,000,000 chain length, 500 subsampling frequency and 300,000 burn in length values. It took about 2 hours and 30 minutes and gave me this result.

Parameter estimates look pretty good and the trace graph has that caterpillar thing shown on the graph.

This is my tree view. My tree included multiple clades with the support value that is greater than 0.85. And I think that these individuals within in each clade are found in the same population.

I think that these concatenated EPIC markers provided enough resolution to distinguish populations phylogenetically  because we, as a class, gather 4 different primers to make 4 different nucleotide alignment making it more precise and accurate than using just one primer. My analysis couple weeks ago was on JP 5334, which is the weird one, so I don’t know if it came out correctly, but it looks like I have a little more resolution than the single marker analysis on JP 5334 couple weeks ago.

11/7/18 Wednesday Plant DNA PCR-ISSRs

Professor Paul ran our Genomic DNA of the samples from last class, several samples were successful and some weren’t.

In this lab, we used two interspersed-simple-sequence-repeat (ISSR) primers in PCR reaction. We used samples of Lupinus arboreus samples from last years class. It was the same species that we saw in our field trips.

First, I made 1:10 dilutions of 5 Lupinus arboreus samples. Three 1.5 mL tubes with unique ID. I pipette 45 micro liters of water and 5 micro liters of each samples into each of the three 1.5 mL tubes. Then I put total of 15 tubes into a rack so that every table gets the diluted samples.

After everyone was done with dilution, I grabbed 15 diluted tubes: PRL 01,PRL 02, PRL 03, PRL 04, PRL 05, PRM 01, PRM 02, PRM 03, PRM 04, PRM 05, PHT 01, PHT 02, PHT 03, PHT 04, and PHT 05. I then grabbed two 0.2 ml 8-tube strips (PCR tube strips) that I will be using for the 15 reactions per marker. I wrote sample ID, primer number (17898) and my initials. I then using filter tips, I pipette 1 micro liter of my first template DNA (dilution) into the first tube of my PCR strips that I labeled. I continued this step and changed the tips everytime I changed my samples. After I was done transferring 1 micro liters each into 15 PCR strips, I put them on ice.

Kayla V. then created Master Mix for our table. Our Master Mix was based on per 80 reactions so the measurements were ddh2o=1000 micro liters, 10x buffer +mg=240 micro liters, BSA=80 micro liters, dNTPs=160 micro liters, Primer (17898)=20 micro liters, Taq=20 micro liters and my template=1 micro liter. Total of 1520 except the amount of my template DNA.

After she was done creating the Master Mix, I pipette 19 micro liters of the Master Mix and added to each of the PCR strips adding up to 20 micro liters into each PCR strips. Every time I added the Master Mix, I mixed the solution by gently pipetting up and down.  After pipetting in 19 micro liters of the Master Mix into each PCR strips (15 time), I closed the lid tightly and put them into the PCR machine.

That was the end of the lab on Wednesday.

10/31/18 Wednesday. In this lab, I used Geneious to code heterozygotes and find polymorphism in the sequences.

Our EPIC marker failed, EPIC failure haha. So we used last year’s data, which were 5334 forward and reverse reads (25 reads of Mimulus cardinalis and outgroup of Mimulus lewisii).

First, I created forward and reverse sequences folder and dropped the reads into the file. I downloaded the reads from Canvas. Next, I selected all the sequences, both forward and reverse reads, and build an alignment using Muscle (default setting). Then, I looked at my nucleotide alignment and cut the ugly reads and looked for polymorphism and heterozygotes. I found many polymorphism and misreads, but about 10 true heterozygotes. Only 4-5 were true heterozygotes and polymorphic. I recoded the true heterozygotes with the appropriate ambiguity code from the IUPAC Ambiguity Code list I have been given in the beginning of the class. After I was done editing, I saved and applied the changes. Next, for each Forward and Reverse pair, I De Novo Assembly them and edited the assembly document so that it has good reads and correct Ambiguity Code. I repeated the same step from another pairs except the outgroup (TG0248) and forward read that did not have a pair (JP 1132). Then, I selected all the Assembly files and generate consensus sequence, and create sequence list. I then extract sequences from list and created a subfolder named 5334 Consensus Sequences. This will move all my Assembly documents into this subfolder. I then manually moved my outgroup reads and JP 1132 reads. Next, I selected them all and choose Edit, Batch Rename and click Remove button and put 33 to remove all the characters except the sequence name for the selected documents. However, I had to edit the name of the outgroup and JP 1132 by hand. After that, I selected all and created alignment using Muscle. I opened the alignment and did some trimming.

AT HOME – INFER A BAYESIAN TREE OF THIS SINGLE LOCUS ALIGNMENT:

I inferred a Bayesian phylogenetic tree using  5334 nucleotide alignment and used the outgroup TG0248 and ran it for about 1hour and 30minutes. This is what I got after the run:

Left is the parameter estimates and right is the trace (good fuzzy caterpillar).

Tree 5334 consensus sequence-2krc97c

If screenshot the screen and paste it here, it came out blurry, so I just created a link to the Bayesian phylogenetic tree of 5334 consensus sequence.